JPH02468A - Method for introducing extraneous dna into lactobacillus - Google Patents

Abstract

PURPOSE:To directly introduce a desired DNA into the cell of lactobacillus by adding an extraneous DNA to a suspension produced by suspending lactobacilli in a buffer solution and applying pulse electric current to the mixture. CONSTITUTION:Lactobacillus cells (e.g., lactobacillus casei) of the prophase of logarithmic growth phase are suspended in a buffer solution (e.g., sodium phosphate buffer solution) of pH 5.5-9 and mixed with >=0.01mug/cuvette of extraneous DNA (e.g., plasmid pAMbetaIN 0.1) and, as necessary, <=30wt.% of polyethylene glycol. The suspension is introduced into a vessel having a pair of plate electrodes and a high-voltage pulse current is passed through the suspension at 0-40 deg.C to effect the electroporation. The objective transformant is selected from the treated product by conventional selection process.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention is directed to the use of Lactobacillus spp.
foreign DNA in the cells of bacteria (Lactobacillus) (US J4)
Regarding the method of introducing A.

More specifically, the present invention relates to an electroporation method (
1! The present invention relates to a method for obtaining transformed Lactobacillus by applying the pneumatic perforation method to Lactobacillus and introducing foreign DNA into the cells of the S. macerate.

[Conventional technology]

Many attempts have been made to date to introduce foreign genes (DNA) into microorganisms and cause the host microorganisms to express the genetic information of the foreign genes. Various methods have already been proposed for obtaining microorganisms, ie, transformants, into which this foreign gene has been introduced.

However, the introduction of foreign genes has its own conditions and problems depending on the type of microorganism, and even if a method is applicable to a specific microorganism, it may not be applicable to other microorganisms. There are also many.

For example, methods for introducing foreign genes into E. coli include methods using CaCl2 and methods using polyoxyethylene, and the protoplast method is widely known for Bacillus subtilis and yeast, but for Lactobacillus No matter which of the above methods is used, it is difficult to introduce foreign DNA into cells, and it has been desired to develop a method for directly introducing foreign DNA into Lactobacillus cells. .

Lactobacillus is an important bacterium that is widely used in industrial fields such as the production of dairy products such as yogurt, lactic acid bacteria drinks, and cheese, the production of feed, and the production of lactic acid by fermentation. If it were possible to artificially modify and breed seeds like this, the industrial benefits would be extremely large, and therefore, genetic engineering methods as a means of breeding should become a very important means from this perspective. .

However, as mentioned above, foreign DN in Lactobacillus
A direct method for introducing A has not been established, and only a few methods have been used to introduce A, W.

Shrago at, al, Appl, Envir
on, Miorobiol, .

52, pp. 574-576 (1986)) and a method of introducing DNA after preparing protoplasts (Japanese Unexamined Patent Publication No. 60-50686) are known to date. This consulging method requires two types of bacteria, a DNA donor and a DNA recipient, and is complex. Not only does it require random mating conditions, but it also requires selective DNA acceptance of the desired gene. It is almost impossible to introduce it into bacteria. Therefore, at this stage, repeated trial-and-error experiments are required, and the reproducibility of the results is poor, making it difficult to call this a reliable method. In addition, in the protoplast method, it is necessary to go through the protoplast formation step of Lactobacillus, and if protoplast formation is insufficient, the introduction efficiency will be low, and if protoplast formation progresses too much, the survival rate of bacteria will decrease. In addition to the problem of 1
This is not an established method for DNA introduction, as there are some unresolved issues regarding the protoplast preparation and restoration methods. By the way, recently foreign DNA has been introduced into mammalian cells.
Electroporation method was proposed as a method to introduce this.

In this method, a suspension of cells and the DNA to be introduced are placed in a container with a pair of iK poles, and a high-voltage pulsed current is passed through the cells suspended in the suspension to change the cell membrane (for example, by physically The idea is to introduce foreign DNA into the cell through this altered membrane. In other words, this electroporation method attempts to utilize increased permeability of substances based on local temporary disruption of cell membranes, and has so far been used to introduce exogenous DNA into large animal and plant cells. This technology has almost reached the stage of being established. Attempts to apply this electroporation method to microorganisms have already been made to protoplasts of yeast and bacteria (B, oereus) [Review by Hiroko Inaba and two others, "Gene Introduction into Mammalian Cells by Electroporation", Protein Nucleic Acid Enzyme vol.1.32.應1 (1987)1
[see pages 0 to 21], however, there are no known reports reporting the applicability of this method to Lactobacillus.

[Problem that the invention seeks to solve]

From the above, it is clear that foreign DNA is present in Lactobacillus cells.
Although there is a need for a method for directly introducing A, the above-mentioned known methods for introducing exogenous DNA into cells have not been applicable.

Therefore, there was a need to develop a new method for introducing exogenous DNA into cells using this lactic acid frame.

[Means for solving problems]

In view of the current situation, the present inventors have conducted extensive research in order to overcome the problems in genetic manipulation of Lactobacillus bacteria and to establish a useful technique for introducing foreign DNA into the cells of this bacterium.

As a result, by applying the electroporation method to Lactobacillus bacteria, we succeeded in directly introducing the desired DNA and completed the present invention.

That is, the present inventors cultured Lactobacillus bacteria, collected bacterial cells, suspended them in buffer KWIA, added and mixed exogenous DNA, and applied a pulsed current to this suspension to remove exogenous DNA. They discovered that it is possible to directly introduce DNA into lactate rod cells.

Therefore, the present invention provides a method for injecting exogenous DNA into floating cells of Lactobacillus.
This method is characterized by applying a pulsed current in the presence of lactobacillus and directly introducing foreign DNA into the cells of lactobacillus.

Furthermore, the present invention is characterized in that polyethylene glycol or the like is present when the pulsed current is applied in the presence of the above-mentioned exogenous DNA.

Lactobacilli that can be transformed according to the present invention belong to all species included in the genus Lactobacillus, and a representative example is Lactobacillus casei.
lus oagei), lacto, ztilus acidophilus (Laotobaoillus aaidoph
illus), Lactobacillus bulgaricus (Laot
obaoillusbulgaricug), Lactobacillus upp.
1actis), Lactobacillus helveticus (
Lactobaoilug helvetiaua
), Lactobacillus saliparius (Laatobao
illusgali marius), Lactobacillus plantarum (Laotobaillus + p
lantarum), Lactobacillus fermentum (Laotobacillus fermentum)
), Lactobacillus cellobisus (Laotob
aoilluscellobioaug ), Lactobacillus delpulchie (Lactobacillus +
delbrueokii), Lactobacillus puhineri (Lactobacillus buohnsri)
), Lactobacillus plebis (Laatobaci)
llua brevig).

Although the method of the present invention is applicable to lactic acid bacteria in the induction phase, growth phase, and stationary phase, it is preferable to use bacterial cells in the early logarithmic growth phase. This logarithmic growth phase is the period when bacteria are inoculated into a medium and cultured. 1 The period when the bacteria that grows the most after the initial period (lag phase) when the bacteria grows the most (log phase)
This refers to the time when the relationship between the logarithm of the number of bacteria (or turbidity) and the culture time becomes a linear relationship. If a pulse current is applied to bacteria at a stage other than this period, the efficiency of introducing foreign DNA will be low.

During the first half of the logarithmic growth phase, which is a suitable period for the introduction of foreign DNA, Lactobacillus bacteria are cultured in an appropriate medium, and the logarithm of the turbidity of each cultured bacteria is plotted against each culture time (growth curve), which is constant. It is defined as the time when the turbidity falls within the range of . For example, when Lactobacillus casei strain IAM1045 is cultured in Briggs (Br1ggg) medium, this corresponds to the time when the turbidity reaches A600 = about 0.05 to about 0.30.

The bacteria thus cultured are collected by appropriate means, such as centrifugation. In this case, separation methods that damage the bacterial cells or reduce their survival rate must be avoided.

The thus collected Lactobacillus bacteria are dispersed and suspended in a buffer solution.

As the buffer used for this suspension of Lactobacillus, known buffers can be used, and those with a pH range of 5.5 to 9 can generally be used, such as sodium phosphate buffer, Tris salt, etc. 1! i! buffer or these a
Adding sucrose to the buffer solution can also result in bτ.

Although it is generally preferable that the number of bacteria in this Lactobacillus suspension be to some extent high, it is sufficient within the range that useful transformants can be obtained.

To this suspension of 14J acid bacilli, foreign DNA to be introduced into the cells of the bacteria is added. Generally, the larger the attachment device, the higher the probability of introduction into cells. Usually 0.0
It can be carried out if it is 1μ9/cuvette or more.

The DNA added here includes all the DNA that can be used for the transformation of lactate, and includes, for example, 2 to 3K DNA.
They can range from relatively low-molecular beaks of bN degrees to those with high molecular weights of 50 to 100 Kb or more. Specific examples of such DNA include ν諷β1 (boiled 1 and A2); pEIL.

1003 and 1) BL1027 (patent application 1986-29!
Prayumide described in No. 1641)
;p0194 and the like.

The suspension containing exogenous DNA is then subjected to electroporation.

In this method, the above-mentioned suspension is generally introduced into a container with a pair of flat plate electrodes, and electroporation (
Ventilation perforations) are performed.

Many devices for this electroporation are commercially available, such as Bio-Rad (Bio R
GonePulsar manufactured by at )
; trade name), GTgl manufactured by Karasu Seisakusho, Beacon 2000 manufactured by Beacon Corporation, Pd5Ino of the United States, Zarapar manufactured by ILJ, etc. can be used in this method.

In this method, the transformation efficiency can be further increased by performing electroporation in the coexistence of, for example, polyethylene glycol.

As polyethylene glycol, for example, polyethylene glycol 2000.4000.6000 and 200 o'yi;' can be used. Moreover, the higher the polyethylene glycol concentration, the higher the transformation efficiency is generally shown. Preferred efficiencies are generally achieved at concentrations up to about 50% (% polyethylene glycol serum in suspension), although higher concentrations are possible.

In addition, in the method of the present invention, ygc is added to the suspension to be energized.
Inorganic substances such as t2, CaCl2, MnC42, etc. may be present, but it is preferable that the amount present is small.

The Lactobacillus bacteria subjected to electroporation in this manner are then subjected to a known separate process A, and the resulting transformants are selected. Transformants are selected based on, for example, resistance to various antibiotics, special ability to assimilate sugars, and requirement for amino acids. Genes with these abilities have already been included in the DNA used in the present invention.

Next, this invention will be explained in more detail with reference to Examples. Note that these Examples are disclosed to explain the present invention, and the present invention should not be construed as being limited by these descriptions.

Example 1 A Priggs (Br1gg5) medium having the following medium composition was prepared by a conventional method.

Medium composition Nidomad juice extract 40〇-Glucose 209 Starch 0.59 Yeast extract [manufactured by Dirco]
6 Kupori is 1s [Daiku Nutrition Co., Ltd.]
y Sodium glutamate 29
CH3COONJL -3H2O109KH2PO44
9 NaCt59 Distilled water Added until the total amount was 1000 tILt pH = 6.8 Tomato juice extract was prepared by adding an equal amount of distilled water to commercially available tomato juice (manufactured by Del Monte) and boiling at approximately 60°C. Heat for 1 hour, then further heat at 100℃ for 5
After heating for a minute, the mixture was cooled and the water-insoluble material was prepared from door to door.

The above medium is used by adding a solution prepared by dissolving 200 q of cysteine and 3.49 ml of sodium asfulupate in 10 ml of distilled water.

A seed culture of Lactobacillus casei (L, casei) IAM1045 strain 0.2-, which had been statically cultured overnight at 67°C in the obtained medium, was inoculated into the above medium 10-, and cultured at 37°C. Culture solution 20- to early logarithmic growth phase (A6oo
=0.25) Centrifugation to collect bacterial cells: 5000
r, p, m, 10 min), this was mixed with room temperature buffer I (7 mu
Sodium phosphate buffer H7,0), 1mM Mg
After washing with Cl2 and 0.4 M sucrose], 25 d of the same buffer IO was added to suspend the bacterial cells. The resulting suspension 0.1-140% polyethylene glycol 600° aqueous solution 0.3 μl and 14 μl of the plasmid carrying the erythromycin resistance gene I) AMβ1
After mixing the ingredients and storing for 10 minutes,
.

Gone Pulsar manufactured by R&(1)
Electricity was applied at room temperature under the condition of 5kV/1-1R% 1μF using ar). After electricity was applied, it was left standing for 10 minutes, and then heated to 15,000 r, p.
The cells were collected by centrifugation at , m, for 2 minutes.

The collected cells were suspended in 11 nt of the above medium and kept at 37°C for 2 hours. Next, 0.3-ml of this suspension was applied to an erythromycin-containing Br1gg5 agar medium and cultured for one week at 37°C under anaerobic conditions, resulting in 161 erythromycin-resistant colonies.

A plasmid was extracted from the obtained erythromycin-resistant colony, and its plasmid size and restriction enzyme cleavage pattern were compared with those in the case of 1) AMβ1m1 described above, and were found to be the same. This is Kazeie, deception 1045
Strain K 1) Indicates that AMβIA1 was introduced.

In addition, 1) the above steps were repeated without adding AMβ1ya1, but no erythromycin resistant colonies were obtained.

In addition, in the above examples and the following examples, extraction and confirmation of plasmids from erythromycin M colonies (abbreviated as rtffi/R colonies) were performed according to the following procedure.

Plasmid extraction Br1gg containing 1μ9/me of erythromycin
5 Plant a loopful of Em colonies in 5 ml of medium,
A seed culture solution was obtained by statically culturing at 37°C overnight. Br1ggg medium 100d containing 3 μl/- of erythromycin
K. This seed culture was inoculated and cultured overnight at 37°C. Next, 100 ml of the obtained culture solution was poured into Briggs medium 90 (l) containing 3 μ9/- of erythromycin.
d and statically cultured at 67°C for 4 hours. Centrifugation to collect bacterial cells: 6000 r, p! I1. .

TE8 (50mM TriB-
CL+50mM Na1j+5mM gDTA; pH8
, 0) 50- and centrifuged again to collect the bacterial cells. A 25% sucrose aqueous solution (25% sucrose a-/C15nM Trim-CL-1m
M RDTA; pHs, o) Suspended in a-, and added with lysozyme water bath solution (lysozyme 10WIg10.25M
Trim-CL1td; pH8,0)2-
was added and kept at 37°C for 1 hour.

Thereafter, it was cooled to 0°C, and 0.25M BDTA (pH 8
, 0) was added, then 1 g of 10% SDB (sodium lauryl sulfate) was added, and the
It was left standing for 0 minutes. Add 5 M N&(1'L to 5.6
- Added, stirred gently and left at 0°C for 1 hour,
A supernatant was obtained by centrifugation (24000 r, p, m, 30 minutes, 4°C). Add an equal amount of phenol-C to this supernatant.
IAA a solution (TE (10mM Trim-Ct+1
mM EDTA; pH 8,0) saturated phenol-volume and chloroform:amyl alcohol (24:1
) Mixture with 1 volume of mixed solution] and shake, 9000 r
, p, m for 10 minutes to obtain a supernatant. This operation was repeated two more times. Add the same amount of CIAA to the resulting supernatant.
was added, shaken, and centrifuged at 9000 r, p, m for 10 minutes to obtain a supernatant. 3 M of this supernatant K[l#
After adding CH3COONa aqueous solution and 2.2 times ethanol and leaving it at -80°C for 1 hour, 9000 r
, p, m, for 10 minutes. The obtained precipitate was
The plasmid band was collected by washing with 0% ethanol, drying, and cesium chloride ultracentrifugation (99,000 r, p, ys, 16 hours). This was desalted using a Sephadex G-25 column, extracted three times with phenol, washed with ether, then precipitated with ethanol, and the precipitate was washed with 70% ethanol. After drying,
The obtained cilasmid was dissolved in distilled water and subjected to the following tests.

Confirmation of plasmid Plasmid size 2
The plasmid extracted from the colony and the EXAMβ1m1 plasmid are subjected to agarose gel electrophoresis to observe their mobility, and when they show the same mobility, they are evaluated as having the same size.

2. Restriction enzyme cleavage pattern: plasmid extracted from the E♂ colony obtained as above and 1) A
The Mβ1m1 plasmid was digested with restriction enzymes (gcoRI, pv
cleaved with u, [, inhibitor II, Kpm 1, etc.),
Perform agarose gel electrophoresis to compare the cut fragments,
If the number of cleavage fragments and fragment size of both are the same, it is determined that the two plasmids are the same.

It should be noted that the operation of Example 1 described above was carried out in the early phase of the logarithmic growth phase (A6
Buffer II (7 m
M) Lis-HCl buffer (p) 48.0), 1mM, MgC
12 and 0.4M sucrose], and 40 erythromycin-resistant colonies were obtained.

Example 2 Coupling conditions of scene pulsar: 1.25 kV 10n,
The method of Example 1 was repeated except that 24 PF was used.

Sixty-eight erythromycin-resistant colonies were obtained.

A plasmid was extracted from this colony and used as I) AM
When compared with β1 short 1, the plasmid size and restriction enzyme cleavage pattern matched. L, Kazei IA
pAMβ1-1 was introduced into the MIQ45 strain.

Furthermore, although the above steps were repeated without adding PAMpIAl, no erythromycin-resistant colonies were obtained.

Example 6 The method of Example 1 was repeated by changing the collection time of the bacterial cells in Example 1, that is, using bacterial cells at different times during the growth of the bacteria.

The results are shown below.

A400: Indicates absorbance (0, D,) at 600 nm.

A plasmid was extracted from the obtained colony in the same manner as in Example 1 and confirmed to be pAMβ141.

Example 4 Cells with A6oo = 0.2 were used instead of the collected cells in Example 1, and instead of pAMβ1A1, a part of the DNA of pAMβ1A1 was deleted. Cilasmid I) AMβ1 Tomb 2
1 μl of DNA was added and the method described in Example 1 was repeated.

Five erythromycin resistant colonies were obtained per agar plate.

When plasmids were extracted from each of the erythromycin-resistant colonies and compared with pAMβIA2, the sizes of the plasmids and restriction enzyme cleavage patterns matched, indicating that 1) AMβ1A2 was introduced.

Example 5 In the medium of Example 1! A seed culture of Lactobacillus caseienia 1045 strain 0.2-, which had been left to stand overnight at 17°C, was inoculated into the above medium 10-, and then left to stand to be cultured at 37°C. Collect A6oo = 0.2 bacterial cells from 80 g of culture solution and centrifuge: 3000 r, pom, 10 minutes, 4°C), add this to 4°C buffer 1:81!1M) Lis-HCl buffer ( pH8
, 5) and 0.4 M sucrose] for 10 d, 0.75 sj of the same buffer was added to suspend the bacterial cells. 0.1 ml of the resulting suspension, 12μ9 of the plasmid β1 containing the erythromycin resistance iIl gene.
and 40% polyethylene glycol (pwa-60Q
a) Mix 0.3 d of aqueous solution and heat at 0°C for 10 minutes.
”//α, 1 μF, current was applied at 0°C. After energization, 0
℃ for 10 minutes, centrifugation (15000 r, p, m
, 2 minutes, 4°C), and the bacterial cells were collected.

The collected bacterial cells were suspended in the above medium 1- and cultured at 57°C for 6 hours. Next, this suspension α5txt was applied to an erythromycin-containing Br1ggg agar medium and cultured for one week at 37° C. under anaerobic conditions, resulting in 4900 erythromycin-resistant colonies.

A plasmid was extracted from this colony in the same manner as in Example 1 and confirmed to be pAMβIA1. Moreover, when no plasmid was added, no erythromycin-resistant colonies were obtained.

Example 6 Polyethylene glycol (pgo60
The method of Example 5 was repeated by changing the leakage of 0.00) to 0.5.10.15.20.25% (PEG i i% in the coupled suspension). The results obtained are shown below.

A plasmid was extracted from the resulting colony in the same manner as in Example 1 and confirmed to be pAM/31A1. Moreover, when no plasmid was added, no resistant colonies were obtained.

Example 7 In place of the 12 μl of plasmid pAMβ in Example 5, 0.4 μl was used and polyethylene glycol (p
The method of Example 5 was repeated using the following types of gG): The results obtained are shown below.

PE02000 480PEG4000
72PE06000
68PEG20000 109
Next, in the same manner as in Example 1, a plasmid was extracted from the resulting colony and confirmed to be pAMpIAl. Moreover, when no plasmid was added, no resistant colonies were obtained.

Example 8 K used in Example 5 instead of the casei IAM1045 strain
L. acidophilus S8/27 strain (FEBM8551
) (PEC) 6000 used) Andori, Kazei JCM
The method of Example 5 was repeated using the IC153 strain (using PE() 2000) and using 5:icv/m and 1 μF as the current application conditions.

In the case of L. acidophilus SS.27 strain, three erythromycin-resistant colonies were obtained;
In the case of strain M1053, 27 erythromycin-resistant colonies were obtained.

Next, a plasmid was extracted from the obtained colony in the same manner as in Example 1, and 1) it was confirmed that it was AMβIA1. Furthermore, no resistant colonies were obtained when no plasmid was added.Example 9 In place of the L6 casei IAM1045 strain used in Example 5, L. acidophilus 8B.27 strain and L. acidophilus strain IAM1045 were used.

acidophilus strain ATCC 4356, and plasmid pCi94 instead of 1 for plasmid pAMβ1.
Also, instead of the Br1gg8 agar medium containing erythromycin, Br containing 5μ/- of chloramphenifur was used.
Using igfl agar medium, 5kV/cm, 1μF
The method of Example 5 was repeated under the energization conditions.

In the case of L. acidophilus SS 27 strain, chloramphenicol resistant colony mosquito of 4@? I I-)Re, also L
In the case of Acidophilus ATCC4356 strain, 12 chloramphenifur-resistant colonies were obtained.

Next, in the same manner as in Example 1, a plasmid was extracted from the obtained colony and confirmed to be pc194.

Example 10 Centrifugation to collect bacterial cells from a seed culture of Lactobacillus casei strain IAM1045 incubated overnight at 37°C in the medium of Example 5: 3000 r, p, m, 10 minutes,
4'C) After washing this with a 4°C buffer [13 mM Tris-HCl buffer (pH 8,5) and 0.4 M sucrose 10-], add 0.75 d of the same buffer and remove the bacterial cells. The WIA was clouded. The resulting suspension 0,1 m
1, 12 μl of plasmid β1A having an erythromycin resistance gene and 0.3 μl of a 40% polyethylene glycol (PE()-6000) aqueous solution were mixed;
After standing at 0°C for 10 minutes, using a scene pulser manufactured by Bioland, under the conditions of 5 kV/cm and 1 μF,
Electricity was applied at 0°C. After electricity was applied, the mixture was allowed to stand at 0°C for 10 minutes, and centrifuged (15000 r, p, m, 2 minutes, 4°C) to collect the bacterial cells.

The collected bacterial cells were suspended in the above medium 1dK and cultured at 37°C for 3 hours. Next, 0.5-ml of this suspension was applied to an erythromycin-containing Br1gg11 agar medium and cultured for one week at 37°C under anaerobic conditions, resulting in 6 erythromycin-resistant colonies.

A plasmid was extracted from this colony in the same manner as in Example 1 and confirmed to be pAMpIAl. Moreover, when no plasmid was added, no erythromycin-resistant colonies were obtained.

Example 11 The method of Example 5 was repeated with various changes in the timing of bacterial collection. The results are shown below.

A6oo = absorbance at 600 nm (0, D, )
A plasmid was extracted from the obtained colony in the same manner as in Example 1, and it was confirmed that the plasmid was I) AMpI A1.

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Claims (1)

[Claims] 1) Lactobacillus is suspended in a buffer solution, and foreign DNA is added to it.
A method for introducing exogenous DNA into Lactobacillus cells, which comprises adding and mixing the following, and applying a pulsed current to this suspension. 2) The method according to claim 1, wherein the Lactobacillus is in an early logarithmic growth stage. 3) The method according to claim 1 or 2, further comprising adding polyethylene glycol to the suspension and applying a pulsed current in the presence of polyethylene glycol.